Project Summary/Abstract: Kidney stones (KS) affect ~1 in 5 men and ~1 in 11 women, are very painful & costly (>$10B annually), and are associated with CKD and ESRD. High recurrence rates (50% in 5 years) indicate that current interventions are inadequate and alternative therapies are needed. Most KS are composed of calcium oxalate, and small increases in urine oxalate enhance the risk for stone formation. Lower urinary calcium oxalate supersaturation definitively reduces KS formation, but unfortunately there are no drugs that reduce urinary oxalate excretion. The gut bacterium Oxalobacter formigenes (Of) induces colonic oxalate secretion and reduces urinary oxalate excretion via an unknown secretagogue. Given the difficulties with recolonization, Of alone is not therapeutically feasible and underscores the need to identify the secretagogue. We identified Of-derived factors secreted in its culture conditioned medium (CM) that stimulate (>2.8-fold) oxalate transport by human intestinal Caco2-BBE (C2) cells through PKA activation and stimulation of the oxalate transporters SLC26A6 (A6) and SLC26A2 (A2). In vivo, rectal administration of CM reduced urinary oxalate excretion >32.5% and stimulated colonic oxalate secretion >42% in hyperoxaluric mice, reflecting the therapeutic potential of these factors. We have identified Sel1 proteins as the major Of-derived factors and they similarly stimulate (1.4-2.4-fold) oxalate transport by C2 cells via PKA and the A2/A6 transporters. We also identified small peptides (P8+9) within a Sel1 protein that stimulate (>2.4-fold) oxalate transport by C2 cells. P8+9 also stimulated oxalate transport by human sigmoid colon (1.8-fold), distal colon (1.7-fold), and ileum (2-fold) organoids (ex vivo intestinal epithelia models fully mimicking the in vivo responses), confirming that P8+9 work in human tissues and that they will likely stimulate oxalate secretion in human colonic and ileal epithelia in vivo. Based on these findings I will test the hypotheses that specific P8 and P9 subdomains mediate colonic oxalate secretion and potential therapeutic motifs can be identified, and that P8+9 act via cell surface receptors and specific signaling pathways to activate the involved oxalate transporters (including A2, A6, and perhaps others). The following specific aims will be pursued: 1. Identify the P8 and P9 peptide subdomains that stimulate oxalate transport in C2 cells and human organoids: 1a Identify the shortest functional P8 and P9 peptides subdomains by deleting specific amino acid residues. 1b. Identify the critical amino acid residues in P8 and P9 peptides using a substitution approach. 2a. Identify the involved cell surface receptor(s) in C2 cells. 2b. Define the signaling pathways mediating stimulation of oxalate transport in C2 cells. 2c. Identify the oxalate transporters and the mechanisms by which P8+9 activate them in C2 cells and use A6 null mice to confirm the in vivo role of A6. Achieving the project?s objectives will have therapeutic implications for the prevention and/or treatment of hyperoxalemia and hyperoxaluria, impacting the outcomes of patients suffering from CaOx KS, enteric hyperoxaluria, primary hyperoxaluria, and possibly CKD and ESRD.